This proposal represents the integration and application of concepts and techniques drawn from the disciplines of pharmacology, cell biology, and nephrology to the examination of those cellular processes and mechanisms available for the selective delivery of drugs, prodrugs, or drugs linked to carrier molecules to the various subcellular components of the proximal tubule cells. The objectives of this proposed study are 1) to determine the extent of receptor-mediated endocytosis by the various regions of the plasma membrane of the proximal tubule cell, 2) to determine the specificity of those receptors, and 3) to investigate the relationship between the specificity of the receptor mediated endocytic process and the subsequent intracellular itinerary of the endosome, receptor and ligand. Using preparations of convoluted proximal tubules, two methods of fractionating the subcellular organelles of these cells, isopycnic centrifugation and free flow electrophoresis, will be compared for their ability to efficiently and quantitatively resolve the organelles possessing their normal compliment of constituents, enzyme activities and specific binding receptors for insulin and molecules carrying the 6-phosphomannosyl moiety. By resolving the subcellular organelles it will then be possible to follow the association of radiolabeled phosphomannans, insulin, albumin, and other substances within defined subcellular compartments of the cells as function of prior drug treatment and the specificity of the internalized ligand. The role of the baso-lateral and brush border regions of the plasma membrane in the initial receptor-mediate endocytosis and subsequent receptor recycling process will be examined by tubule microperfusion techniques. A rigorous comparison of the various techniques available for cell fractionation is a timely response to the expanding needs, not only of the cellular pharmacologist, but those interested in the subcellular distribution of various biological processes as a function of disease and age.